猴头菇多糖对MDRV感染RAW264.7细胞TLR3/TRIF诱导表达及病毒复制的影响  

Effects of Hericium erinaceus Polysaccharide on TLR3/TRIF-induced Expression and Viral Replication in MDRV-infected RAW264.7 Cells

作　　者：严萍 林绍青 李明慧 孙雪宁[1] 李健[1,2] 黄一帆 吴异健[1,2] YAN Ping;LIN Shaoqing;LI Minghui;SUN Xuening;LI Jian;HUANG Yifan;WU Yijian(College of Animal Science(College of Bee Academy),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福州350002 [2]福建省兽医中药与动物保健重点实验室,福州350002

出　　处：《中国畜牧兽医》2021年第9期3415-3422,共8页China Animal Husbandry & Veterinary Medicine

基　　金：福建省自然科学基金项目(2017J01597);校科技创新专项基金项目(CXZX2017054)。

摘　　要：试验旨在探究猴头菇多糖(HEP)预处理RAW264.7细胞对番鸭呼肠孤病毒(MDRV)复制的影响及其机制,从Toll样受体3/β干扰素TIR结构域衔接蛋白(TLR3/TRIF)信号通路解析HEP在调节MDRV所致的雏番鸭机体免疫抑制中的作用机制并提供体外试验参考。试验设空白对照组(BCG)、病毒感染对照组(VCG)、HEP对照组(HCG)和HEP预防病毒感染组(HPG),RAW264.7细胞在MDRV感染12和24 h后,通过Western blotting检测各组细胞中TLR3、TRIF和肿瘤坏死因子受体相关因子6(TRAF6)的蛋白表达量;病毒感染24 h后,用ELISA法检测各组细胞培养液中肿瘤坏死因子-α(TNF-α)、白介素-10(IL-10)、IL-6、IL-1β、干扰素-β(IFN-β)的含量。TCID50检测结果表明,MDRV病毒的TCID50为103.46。病毒感染后12 h,细胞未出现明显变化;病毒感染后24 h,细胞变圆;病毒感染后36 h,细胞大量死亡。MDRV在RAW264.7细胞中的复制结果显示,在感染病毒12~24 h内,σNS的表达量呈现上升趋势;在24~36 h,σNS的表达量维持在较高水平。Western blotting结果显示,与空白对照组相比,VCG组细胞TLR3、TRIF和TRAF6蛋白的表达量在感染后12和24 h均显著升高(P<0.05),HCG组TRIF蛋白的表达量均显著升高(P<0.05);与感染组相比,HPG组的σNS、TLR3、TRIF和TRAF6蛋白表达量在病毒感染12和24 h均显著降低(P<0.05)。ELISA检测结果显示,与空白对照组相比,VCG组细胞培养液中TNF-α、IL-6、IL-1β和IFN-β的含量均显著升高(P<0.05);与感染组相比,HPG组细胞培养液中TNF-α、IL-6、IL-1β含量均显著降低(P<0.05),IFN-β的含量显著升高(P<0.05)。结果表明,HEP可调节MDRV感染诱导RAW264.7细胞TLR3信号转导通路活化,抑制TLR3信号转导通路下游产物TNF-α、IL-10、IL-6和IL-1β的过度表达,同时上调IFN-β的表达,从而抑制MDRV在RAW264.7细胞中的复制。The aim of this study was to investigate the effect of Hericium erinaceus polysaccharide(HEP)pretreatment of RAW264.7 cells on the replication of Muscovy duck reovirus(MDRV)and its mechanism,and dissect the mechanism of HEP in regulating MDRV-induced immunosuppression in Muscovy ducks from the Toll-like receptor 3/interferon-βTIR domain adaptor protein(TLR3/TRIF)signaling pathway.The trial included blank control group(BCG),viral infection control group(VCG),HEP control group(HCG)and HEP prevention viral infection group(HPG).The protein expression levels of TLR3,TRIF and tumor necrosis factor receptor-associated factor 6(TRAF6)in RAW264.7 cell at 12 and 24 h of MDRV infection was detected by Western blotting.After virus infection for 24 h,the contents of tumor necrosis factorα(TNF-α),interleukin 10(IL-10),IL-6,IL-1βand interferonβ(IFN-β)in the cell supernatant culture medium of each group were detected by ELISA.The TCID50 detection results showed that the TCID50 of MDRV was 103.46.At 12 h after infection,the cells did not change significantly,24 h after infection,the cells became round,and 36 h after infection,a large number of cells died.The replication results of MDRV in RAW264.7 cells showed that within 12-24 h of virus infection,the expression ofσNS increased,and at 24-36 h,the expression ofσNS was maintained at a high level.Western blotting results showed that compared with blank control group,the expression levels of TLR3,TRIF and TRAF6 proteins in VCG group were significantly increased(P<0.05)after 12 and 24 h infection,and TRIF protein in HCG group were significantly increased(P<0.05)after 24 h infection.When compared with infected group,the expression ofσNS,TLR3,TRIF and TRAF6 proteins were significantly decreased(P<0.05)after 12 and 24 h infection in the HPG group.ELISA results showed that compared with blank control group,the contents of TNF-α,IL-6,IL-1βand IFN-βin the culture medium of VCG group were significantly increased(P<0.05).When compared with infected group,the contents of TNF-α,IL

关 键 词：番鸭呼肠孤病毒 猴头菇多糖 TOLL样受体3 信号转导 病毒复制 

分 类 号：S858.32[农业科学—临床兽医学]